402 SAMJ VOL 76 21 aCT 1989
Molecular characterisation
mutation in exon 8 of the
lipoprotein receptor gene
of a low-frequency
human low-density
M. J. KOTZE,
E.
LANGENHOVEN,
L.
WARNICH,
M. P. MARX,
A.
E.
RETIEF
Summary
The prevalence of familial hypercholesterolaemia (FH), an autosomal dominant disease characterised by raised low-density lipoprotein (LDL) cholesterol levels, is at least five times higher in the white Afrikaner population than in most other population groups in the world. A founder gene effect has been suggested to explain this abnormally high fre-quency. Detection of a polymorphic51u I site in the 5' region
of the LDL receptor gene and association of both restriction fragment length polymorphism alleles with FH in Afrikaners, indicated the existence of at least two founder members for the disease in this population. DNA from a hetero-allelic FH homozygote from this South African group has been analysed through genomic cloning and sequencing. The DNA polymor-phic site is caused by a single guanine to adenine transition within exon 8 of the LDL receptor gene and can be used in the determination of haplotype-associated defects.
SAIr Med J1989; 76: 402-405.
Familial hypercholesterolaemia (FH) is a common autosomal dominant disease caused by DNA mutations in the low-density lipoprotein (LDL) receptor gene locus. The prevalence of heterozygous FH appears to be approximately I in 500 among the general population of most countries in the world, but in Afrikaners FH occurs at a 5 - 10 times higher frequency. A founder gene effect has been suggested to explain this abnormally high frequency.I Clinical studies have demonstrated
that tendon xanthomas and premature coronary artery disease are encountered in middle-aged FH heterozygotes, while in the homozygous condition both cutaneous and tendon xan-thomas as well as aortic and coronary atherosclerosis appear before the age of 10 years.2 It is therefore advantageous to identify affected subjects at a young age before they develop symptomatic disease, in order to counsel on diet and drug treatment to reduce their future risk of myocardial infarction.
Ithas been shown that measurement of total or LDL choles-terol levels does not always allow unequivocal diagnosis of FH, especially in childhood,'·4 since these values may fall in the upper range of normal.
The cloning of the LDL receptor gene5has made it possible
to analyse mutations causing FH at the DNA level. A number of different mutations affecting the LDL receptor, including deletions, insertions, missense and nonsense mutations, have
MRC Cytogenetics Research Unit, Department of Human Genetics, University of Stellenbosch, Parowvallei, CP
M.
J.
KOTZE, M.Se. E. LANGENHOVEN, M.se L.WARNICH, M.Se. M. P. MARX, M.Se.,PH.D. A. E. RETIEF, M.Se.,PH.D. Accepted 8 Aug 1989.Reprinr requeststo:Professor A. E. Retief, Dept of Human Genetics. University of Srellen-bosch Medical School, PO Box 63, Tygerberg, 7505. .
been described.6 Langlois er aP have recently shown that
about 6% of all mutations in the UK population represent gene deletions, which can be detected directly using Southern bloning techniques. The majority of defects are, however, the result of point mutations and will require other methods of diagnosis.
The mutational heterogeneity of FH creates a problem in the application of recombinant DNA methods for diagnosis at the genomic level. Itis usually not feasible to screen genomic DNA from a patient who has the defmed clinical syndrome for all the mutations that are known to have occurred in the suspected gene. Detection by methods that specifically screen for the mutational defect by restriction analysis or by differen-tial oligonucleotide hybridisation may, on the other hand, be successful in populations that carry only a small number of mutations. This was shown in the Lebanese,8 French Canadian9
and Finnish10 FH patients, where the spectrum of LDL
receptor gene mutations was narrower, since the populations are more homogenous in genetic terms. This strategy may also be applicable in the efficient characterisation of the mutations causing FH in the Afrikaner population, which has remained isolated through religious belief and cultural bonds.
LDL receptor studies of South African FH homozygotes have shown a predominance of a receptor-defective type of abnormality.ll This is in accordance with the founder gene hypothesis for Afrikaner FH, implying that the majority of patients will manifest the same mut'ltion in the LDL receptor gene. Since the nature and number of molecular gene defects in this population have not yet been defmed and our Southern blot analyses of Afrikaner FH patients have not shown gross structural alterations, we used restriction fragment length polymorphism (RFLP) studies to investigate the founder hypo-thesis. Ten useful two-allele RFLPs of the LDL receptor gene were used for haplotype studies in the Afrikaner population.12
Pedigree analysis has shown the segregation of at least 17
haplorypes in the normal population compared with a predomi-nant association of two of these haplotypes with the disease in the FH subjects. In 70% of FH families studied the defective gene co-segregated with the rare allele of a Nco I RFLp13 (haplotype 2), while the rare allele of aSw I RFLp14 (haplo-type 6) segregated with FH in 20% of these families. This association was further confirmed in 27 unrelated FH homozy-gotes; 24were homozygous for haplotype 2, while 3 showed compound heterozygosity for haplorypes 2 and 6. Since specific mutations may occur in different populations in close associa-tion with single RFLPs or RFLP haplotype panerns, haplotype analysis can serve as an important means of genetic charac-terisation of a population with regard to a. particular mutation causing the disease.
Association of both alleles of the rareSw I RFLP with FH in some of the Afrikaner families studied, first indicated the existence of at least two 'founder' members for the disease in South Africa. Siidhof eral.15 published data on the genomic map of the human LDL receptor gene indicating the cleavage sites for selected restriction endonuCleases. We used these known fragment sizes and DNA double digests with sets of different restriction enzymes to determine the exact sizes generated bySwI. Comparing the fragment sizes found with
SAMT VOL 76 21 OKT 1989 403
those described by Siidhofecal.,ISwe mapped the variableSw I site within exon 8 of the gene and an extra recognition site for the enzyme was found between exons 15 and 16 to produce the common 7,2 kb fragment.14 Accurate mapping of this variable RFLP site is very important, since it can be used to determine haplotype-associated defects in cDNA from hetero-allelic FH homozygotes. To date, the SwI RFLP is the only variable site found within an exon of the 5' region of the LDL receptor gene and true FH homozygotes for this rare haplotype have not been detected for cloning purposes. In this study, using genomic cloning and DNA sequencing techniques, we confIrm that the Sw I RFLP is located withinexon 8 of the gene and is caused by a single base substitution in this region.
contained within a 15 kb Bgl II fragment.15 To clone this
fragment, genomic DNA from a hetero-allelic FH homozygote (FH 8) was restricted with Bgl II, size-fractionated in agarose gels and fragments of approximately IS kb were used to construct a genomic DNA library in the Barn HI sites of bacteriophage lambda L47.1. The library was screened as described under 'Materials and methods'. This procedure yielded a single recombinant bacteriophage that harboured a human DNA fragment of approximately 15 kb. Restriction mapping revealed that this fragment lacked the Sw I site normally found in exon 8 of the LDL receptor gene and thus represented the FH 8 - haplotype 6 allele.12
1 kb ~ EcoRI I
c
T
G A
~~
C
~+
G
A
A
G
BamHI I . Stu IFig. 1. Restriction map of the 9 kbEco RI fragment of the 5' end
of the LDL receptor gene analysed in FH 8. TheEco RI sites used
for construction of the pBR 328 recombinant clone are shown,
together with the Barn HI and Stu I sites in this region. The
absence of aStu I site within the DNA fragment is denoted by an
arrow.
DNA sequence analysis of exon 8
A 9 kb Eco RI fragment from the recombinant bacteriophage was subcloned into a pBR 328 plasmid vector. A restriction map of the resulting human DNA insert in the plasmid is shown in Fig. 1. Sequencing of the denatured double-stranded plasmid DNA using an oligonucleotide primer specific for the 5' end of exon 8 revealed that allele 6 differed by a single base in the 6-base pair sequence recognised and cleaved by Sw 1.
Fig. 2 shows this portion of the exon 8 sequence, where an
Materials and methods
DNA sequencing
Exon 8 of the LDL receptor gene was sequenced by the dideoxy chain termination method using an oligonucleotide primer specilic for the 5' end of exon 8 (5' TTCTCTCTCTT-CCAGATA3'). A double-stranded plasmid, derived from pBR 328 and containing the cloned insert of lambda FH 8 - 30, was denatured with sodium hydroxide and then annealed with the exon 8 primer.19Sequencing was carried out using a
commer-cial kit (Sequenase). The sequence was compared with that of exon 8 of the normal LDL receptor gene.s
Genomic cloning
High-molecular-weight DNA was prepared from a blood sample ofFH homozygote 8 by a Triton X-lOO lysis method.16
This DNA (300 Jlg) was digested overnight at 37°C with Bgl II using conditions recommended by the manufacturers (Boehringer Mannheim). The digested DNA was subjected to electrophoresis in a 0,6% agarose gel together with DNA size markers. Two DNA fractions were eluted from the gelp after which Southern blotting with a 1,05 kbPscI probe, subcloned into pBR 322 from pLDLR-3, was used to identify the frac-tion containing the desired 15 kb Bgl II fragment. pLDLR-3 contains a full-length LDL receptor cDNA and was obtained from the American Type Culture Collection (ATCC). The human DNA (100 ng) was mixed with 0,5 Jlg Barn HI-digested arms of lambda L47.J18 and incubated overnight at 14°C with T4 DNA ligase (Amersham International). The ligated material was packaged in lambda phage particles in
vicroto yield a total of 1 X 106plaque-forming units. Lambda
DNA packaging extracts (Gigapack Plus) were purchased from Vector Cloning Systems.
Approximately 5 X 105plaque-forming units were screened
after plating on E. coli strain WL 95. One recombinant clone was identifIed with the 1,05 kb Psc I clone, encompassing exons 2 - 8 of the normal LDL receptor gene. This clone, designated lambda FH 8 - 30, was isolated after two additional cycles of plaque purifIcation. Restriction endonuclease mapping and Southern blotting indicated that this clone contained the 15 kb Bgl II LDL receptor gene fragment comprising exons 4 -11. The genomic human DNA insert in this phage lacked a polymorphic Scu I site found in exon 8.14 The DNA from clone lambda FH 8 - 30 was digested with Eco RI and a
purifIed 9 kb human DNA insert (Fig. 1) was subcloned into
Eco RI-digested pBR 328 DNA.
Results
Molecular cloning
In the gene for the normal LDL receptor, exon 8 is
Fig. 2. Autoradiograph of a sequencing gel demonstrating the mutation point in exon 8 of the LDL receptor gene. The DNA sequence in the region surrounding alanine 370 is shown, with the single base substitution indicated (arrow) at nucleotide position 1171.
-404 SAMJ VOL 76 21 OCT 1989
adenine replaced a guanine at nucleotide posltlon 1171, changing amino acid 370 from alanine to threonine. The remaining exon 8 sequence from the cloned genomic fragment is similar to that of the normal gene.
Discussion
The gene for the human LDL encompasses 45 kb of DNA divided into 18 exons on the short arm of chromosome 19. 15 In this paper we characterised a mutation in exon 8 of the LDL receptor gene, giving rise to aSw I RFLP. The presence or absence of this site produces two allelic fragments of IS and 17 kb respectively, in genomic Southern blots using cloned cDNA probes. Analysis of DNA isolated from 60 normal, unrelated Afrikaner individuals revealed that the frequency of the common allele (S1) is 0,93 and that of the rare allele(S2) is 0,07. This did not differ significantly from that observed in our FH population (90 al1eles)ZO or in the UK population.zl Cloning and sequencing data from DNA of a hetero-allelic FH homozygote indicated that theSwI polymorphism was caused by a guanine to adenine transition in exon 8 of the LDL receptor gene. This RFLP, the only one so far detected in the 5' coding sequences of the LDL receptor gene, can be used to distinguish the RFLP alleles for determination of the haplo-type-associated defects in cDNAs from hetero-allelic FH homozygotes. To date, a true FH homozygote for the rareSw I allele has not been found for this purpose. Studies are under way to determine the DNA sequence of the remaining LDL receptor gene exons cloned in lambda FH 8 - 30, using exon-specific oligonucleotide primers. This may permit the mole-cular analysis of the genetic lesion which causes FH among Afrikaners carrying the haplotype 6 allele of the LD L receptor gene.
At least four different classes of LDL receptor gene muta-tions can be distinguished based on measurements of LDL receptor activity found on the surface of cultured fibroblasts. These disrupt synthesis, intracellular transport, LDL-binding ability or internationalisation of the LD L receptor.22A variant
of the class 2 mutation that produces receptors that are converted to the mature form at an abnormally slow rate has been identified in WHHL rabbits and in several individuals with homozy¥ous FH,23 including three South African FH homozygotes. 1.24 Previous studies have suggested that altera-tions in cysteine-rich repeats of the LDL receptor gene are likely to disrupt the disulfide pattern of the protein in these regions, causing abnormal movement to the cell surface. 25 Three different FH mutations that disrupt the transpoI:l of the protein to the plasma membrane have been localised to the cysteine-rich repeats. Yamamotoer al.25reported that the gene
mutations in the WHHL rabbits and a patient with FH involve small in-frame deletions in the fourth exons of their LDL receptor genes. A third defect is attributable to a single base substitution in exon 14 that produces a termination codon in the middle of a cysteine-rich sequence.8Recently Esser and
RusselP6 have characterised a fourth mutation in exon 11 of the LDL receptor gene that encodes a transport-deficient protein. In contrast to the earlier defects, this mutation does not reside in the cysteine-rich repeats, but causes a substitution of a valine for a glycine at residue 544. These results suggest that free cysteines are not obligatory for the blocked intra-cellular movement of mutant LDL receptors and that regions of a polypeptide backbone containing glycine residues may also be sensitive targets for mutations that disrupt folding. The characterisation of additional transport-defective LDL receptor mutations in FH may serve to identify folding hot spots involving glycine. 26
The receptor-defective mutation of the 'slow maturation' type was also reported in a family in which there is unusual
longevity and in which FH heterozygotes did not express constant or statistically significant hypercholesterolaemia.27,28 The fact that obligate FH heterozygotes can have normal or near-normal cholesterol levels called our attention to the fact that a mutant FH allele may be expressed in a background of factors that fail to produce substantial hypercholesterolaemia. With this background, we decided to have a closer look at the base pair changes creating theSw I RFLP within exon 8 of the LDL receptor gene. 14 The frequency of the RFLP is very low in both our normal and FH populations, and the recog-nition sequence for Sw I involves a cysteine codon in the LDL receptor gene. We described the possible role of altera-tions in cysteine-rich regions of the LDL receptor gene in the production of transIJort-deficient proteins above. Our sequen-cing data have revealed that the mutation in exon 8 causes a substitution of a threonine for an alanine at residue 370, which does not cause a gross alteration in protein structure. It is thus highly unlikely that this mutation affects the processing of the receptor protein in FH patients. Biochemical studies of recep-tor proteins of cultured fibroblasts from FH patients homo-zygous for the exon 8 mutation and site-directed mutagenesis will be necessary to detect possible abnormalities in protein behaviour.
To date, more than 20 different mutations, which abolish the function of the LDL receptor protein, have been defined at the molecular level. It is likely that, as the number of such mutations analysed is increased, the application of recombinant DNA technology in the diagnosis of FH will become more precise and more universally applicable. This is of particular importance in South Africa, where the high FH frequency is prevalent as a result of a founder effect.
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homozYllotes in South Africa. Br MedJ 1980; 281: 633-636.
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Metabolzc Basis of Inherited Disease.5th ed. New York: McGraw-Hill 1983:
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3. Leonard JV, Whitelaw AGL, Wolf OH et al. Diagnosing familial hyper-cholesterolaemia in childhood by measuring serum cholesterol. Br Med J 1977; I: 1566-1568.
4. Christensen V, Glueck C, Kwiterovich P et al. Plasma cholesterol and triglyceride distributions in 13665 children and adolescents: the prevalence scudy of the Lipid Research CIUlICS program. Pediarr Res 1980; 14: 194-202.
5. Yamamoto T, Davis LG, Brown MS etal. The human LDL receptor: a
cysteUle-nch proteUl With muluple AJu sequences in its mRN A. Cell 1984; 39: 27-38.
6. Russell DW, Esser V, Hobbs HH. Molecular basis of familial
hyper-cholesterolemia.Art~osclerosis1989; 9 (suppl I): 1-8-1-13.
7. LangIOls S, Kastelem HP,. Harden MR. Characterization of six partial dele.rlOns In the low denSity lIpoproteIn (LDL) receptor gene causing familIal hypercholesterolemia (FH). AmJ Hum Genet 1988; 43: 60-68. 8. Lehrman MA, Schneider WJ, Brown MA et al. The Lebanese allele at the
low density lipoprotein receptor locus.J Bioi Chem 1987; 262: 401-410. 9. Hobbs HH, Brown MS, Russell DW et al. Deletion in the gene for the low
denSIty lipoproteIn receptor in a majority of French Canadians with familial hypercholesterolemia. N EnglJ Med 1987; 317: 734-737.
10. AaIto-SetaHi K, Gylling H, Miettinen T, Konrula K. Identification of a deletion in the LDL receptor gene: a Finnish type of mutation. FEBS Leer 1988; 230: 31-43.
11. Coetzee GA, Van der Westhuizen DR. Familial hypercholesterolaemia - a
receptor defect. S AfrJ Cont Med Educ 1984; 2 (March'): 49-56.
12. Kotze MJ,. Langenhoven E, Retief AE et al. Hap10types identified by 10 DNA restnctlon fragment length polymorphisms at the human low density IIpoprotem receptor gene locus.J Med Genu 1989; 26: 255-259.
13. Kotze MJ, Langenhoven E, Dietzsch E, Retief AE. A RFLP associated with
the low density. lipoprotein receptor~ene.Nucleic Acids Res1987; 15: 376.
14. Kotze MJ, Reuef AE, Brink PA, Welch HFH. A DNA polymorphism in rhe human low density lipoprotein receptor gene. S Afr MedJ 1986; 70: 77-79: 15. Siidhof TC, Goldstein JL, Brown MS, Russell DW. The LDL receptor
gene: a mOSaIC of exons shared With different proteins. Science 1985' 228:
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16. Kunkel LM, Smith KD, Boyer SH et al. Analysis of human Y-chromo-some-specific reiterated DNA in chromosome variants. Proc Narl Acad Sci
USA1977; 74: 1245-1249.
17. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory
Manual.New York: Cold Spring Harbor Laboratory, 1982.
18. Loenen WAM, Brammar WJ. A bacreriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene 1980' 20:
SAMT VOL 76 21 OKT 1989 405
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of LDL receptor impedes transport ro cell surface in WHHL rabbits.
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and
proto-dual porphyria
Uroporphyrinogen decarboxylase
porphyrinogen oxidase in
E.
D. STURROCK,
P. N. MEISSNER,
D. L. MAEDER,
R.
E.
KIRSCH
Summary
The urinary and faecal porphyrin excretory profiles of dual porphyria are said to represent the superimposition of those found in porphyria cutanea tarda on those seen in variegate porphyria. To test this hypothesis the enzymes responsible for these conditions, protoporphyrinogen oxidase (variegate porphyria) and uroporphyrinogen decarboxylase (porphyria cutanea tarda) were measured in vitro in haemolysates and
Iymphoblasts of 10 subjects with dual porphyria in order to clarify the enzymatic defects. Mean protoporphyrinogen oxi-dase activity in Iymphoblasts from subjects with dual por-phyria was decreased by 45% from 0,82±0,10 to 0,45±0,09 nmol protoporphyrin/mg protein/h(P
<
0,001). Uroporphyri-nogen decarboxylase activity was also significantly reduced from 0,12±0,05nmoI7-, 6-, 5- and 4-carboxyl porphyrin/mg protein/h in Iymphoblasts from normal subjects to 0,08±
0,02 nmol in Iymphoblasts of subjects with dual porphyria(P
<
0,01). There was a similar 27% decrease in mean uroporphyrinogen decarboxylase activity of haemolysates from the same dual porphyria group (P<
0,01). Mean activity of this enzyme in 5 patients with variegate porphyria did not differ significantly from that in normal subjects. These findings may well provide a rational basis for the abnormal porphyrin excretory profiles found in subjects with dual porphyria.SAir MedJ 1989; 76: 405-408.
MRC Liver Research Centre, Department of Medicine, University of Cape Town
E. D. STURROCK, B.sc.(MED.)HO~S
P. . MEISSNER,B.SC. (MED.) HO'lS
D.L.MAEDER,PH.D.
R. E. KIRSCH,M.B.CH.B.,M.D., F.C.P. (S.A.)
Dual porphyria is a disorder of porphyrin metabolism present in up to 25% of subjects with variegate porphyria. Subjects with dual porphyria exhibit haem precursor overproduction characteristic of both porphyria cutanea tarda and variegate porphyria.I This relates particularly to the raised faecal
excre-tion of isocoproporphyrin and the elevated urinary uropor-phyrin and 7-carboxyl poruropor-phyrin. The clinical features of dual porphyria are similar to those of variegate porphyria. They include cutaneous fragility and photosensitivity as well as a propensity to develop acute attacks consisting of varying combinations of abdominal pain, vomiting, constipation, pain in the back and limbs, abnormal behaviour, tachycardia, hyper-tension and a predominantly motor neuropathy.2 The cuta-neous involvement in porphyria cutanea tarda is identicalto
that of variegate porphyria, but acute attacks do not occur.I
Variegate porphyria is inherited as an autosomal dominant disorder.3 The enzyme primarily affected is
protoporphyri-nogen oxidase.4-6 There are thought to be two forms of porphyria cutanea tarda: a familial form inherited in an auto-somal dominant manner and a sporadic form, the hereditary characteristics of which, if any, remain unclear.H In familial porphyria cutanea tarda uroporphyrinogen decarboxylase acti-vity is decreased in all tissues.9,10 Although it is generally
accepted that only hepatic uroporphyrinogen decarboxr1ase is decreased in sporadic porphyria cutanea tarda/,8,11-1 some workers have shown a decrease in uroporphyrinogen decarbo-xylase activity in erythrocytes of patients with sporadic por-phyria cutanea tarda.14 We measured the activities of both protoporphyrinogen oxidase and uroporphyrinogen decarboxy-lase in subjects with dual porphyria in an attempt to elucidate the mechanism responsible for the porphyrin excretory profiles associated with this condition.
Accepted28 Dec 1988.
Reprint requests to: ProfessorR.E. Kirsch, Depr of Medicine, University of Cape Town, Observarory, 7925RSA.
Material and methods
We studied 10 patients with dual porphyria. Two of the
